Discusión de los resultados, conclusiones y recomendaciones

The SCR protein contains multiple peptide domains and motifs that have been conserved at different taxonomic levels (FIGURE 1.7and FIGURE 1.8). The HOX5 class

homeodomain (HD) and YPWM motif of SCR protein are sufficient for SCRT1 activity and are conserved in all bilaterian SCR orthologs, including the murine SCR ortholog,

FIGURE 1.7 Conserved regions in Drosophila Sex combs reduced protein. The

structure of the SCR protein (417 amino acids) in D. melanogaster, indicating the taxonomic level of conserved peptide motifs and domains. The octapeptide motif, YPWM motif, homeodomain (HD), and KMAS motif are universally conserved in bilaterians. The LASCY, SCKY, PQDL, and NANGE motifs are conserved in all arthropods and protostomes. The DYTQL motif and C-terminal domain (CTD) are conserved insects. The YTPNL, DISPK, and NEAGS are conserved amongst Dipterans. The NDPVT, QSLAS, and VNVPM are found only in Drosophilids. The grey region of the protein indicates amino acid sequence in which conserved regions have not been identified.

FIGURE 1.8 Alignment of representative sequences of bilaterian Sex combs reduced orthologs. There are multiple conserved regions in orthologous SCR proteins, outside of the homeodomain, which are conserved at different taxonomical levels. Sequence

alignments of orthologous SCR proteins are shown for all conserved regions except the HD. Portions of the amino acid sequence in the C-terminal domain are condensed for simplicity, which is indicated by square brackets and the number of amino acids (aa) encompossed in the condensed portion. In this unrooted tree (left), the length of branch lines does not indicate evolutionary time, but rather, is an indication of taxonomic relatedness. Organisms belonging to the same taxonomic classification are grouped together, such that the taxonomic level of conservation of SCR protein regions is emphasized. Species with an asterisk (*) denote organisms, from which, Scr orthologs were isolated for use in the cloning of the reintegration-ortholog vectors. Accession numbers for SCR orthologs are listed in APPENDIX 1.1. A summary of conserved SCR regions can be found in APPENDIX 1.2.Primary protein sequences were aligned using

HoxA5 (PERCIVAL-SMITH ET AL.2013). The HD is the only domain in SCR with an

essential function: it is both necessary and sufficient for DNA-binding. The remainder of SCR protein is composed of a number of small, differentially conserved peptide motifs. The octapeptide and KMAS motif are universally conserved across all bilaterian SCR orthologs; the LASCY motif is conserved in protostome SCR orthologs; the SCKY, PQDL, and NANGE motifs are conserved in arthropod SCR orthologs; the DYTQL motif and C-terminal domain (CTD) are insect specific; the YTPNL, DISPK, and NEAGS are conserved in all Dipteran SCR orthologs; and the NDPVT, QSLAS, and VNVPM conserved only in Drosophila SCR orthologs (CURTIS ET AL.2006;PERCIVAL- SMITH ET AL.2013;SIVANANTHARAJAH AND PERCIVAL-SMITH 2015). Previous analysis suggests some of these motifs may contribute to SCRlab activity; and from this functional analysis of SCR, it is suggested that three key events occurred during the evolution of insects: i) SCR acquired the negative regulatory DYTQL motif and CTD; ii) SCR acquired the activity to determine labial identity; and iii) PB expression shifted posteriorly to assist in the switch from the bilaterian conserved SCRT1 activity to the insect specific SCRlab activity (PERCIVAL-SMITH ET AL.2013).

A functional dissection of Scr resulted in two key discoveries: (1) Scr exhibits differential pleiotropy, and (2) all of the conserved SCR protein motifs – not including the HD, octapeptide, or CTD – may represent plastic sequence elements, called short linear sequence motifs (SLiMs; SIVANANTHARAJAH AND PERCIVAL-SMITH 2014,2015).

Differential pleiotropy is the observation that short, independently acting peptide elements each make small, additive tissue-specific contributions to SCR activity (CARROLL 2005;SIVANANTHARAJAH AND PERCIVAL-SMITH 2009;SIVANANTHARAJAH AND PERCIVAL-SMITH 2014).In a functional analysis of Scr, hypomorphic Scr alleles

were ranked from weakest to strongest Scr phenotype in three tissues: the sex combs bristles, the proboscis, and the larval salivary glands (SIVANANTHARAJAH AND PERCIVAL-

SMITH 2009).If every region in SCR was uniformly required in all tissues, the same

allelic series would be expected for each tissue. However, there was a differential

requirement of the octapeptide, DYTQL, NEAGS, YPWM, and CTD in all three tissues. This study concluded that SCR functions are distributed throughout the protein in small, additive functional motifs that are important, but not essential for SCR activities

(SIVANANTHARAJAH AND PERCIVAL-SMITH 2009).Some of these SCR motifs may be

SLiMs, which are small motifs of 3-10 amino acids that act as effector binding sites with widespread cellular function (SIVANANTHARAJAH AND PERCIVAL-SMITH 2015). Many

SLiMs in SCR appear to have seemingly non-essential functions. The YPWM motif, for example, is important but not essential for SCR function as it can be deleted with

minimal phenotypic effect. The expression of Scr3, a hypomorphic allele, which has a YPWM change to YLWM, has only a small effect on salivary gland development in Drosophila (JOSHI ET AL.2010;SIVANANTHARAJAH AND PERCIVAL-SMITH 2009).

Differential pleiotropy is a genetic observation which suggests that some SCR protein motifs are important, but not essential, for protein function and it provides a mechanism to facilitate Scr gene evolution by reducing the pleiotropy of a mutation (HITTINGER ET AL.2005;MERABET ET AL.2011;SIVANANTHARAJAH AND PERCIVAL-SMITH 2015). SCR plays a crucial role in conferring labial and prothoracic segmental identity in Drosophila. Previous analysis of Scr orthologsindicate that SCRT1 activity is conserved in Scr orthologs. SCR-dependent prothoracic phenotypes, such as the T1 beards and male sex comb, generated by SCRT1 activity can be induced by ectopic expression of Scr orthologs, like murine HoxA5 (ZHAO ET AL.1993). However, the emphasis of my

research is to identify the time at which SCRlab activity arose. One proposal suggests that labial activity is insect specific (PERCIVAL-SMITH ET AL.2013). In this case, the rescue of

SCR-dependent labial phenotypes would only be observed with insect Scr orthologs. However, my analysis is not limited to testing this specific hypothesis. It is possible that SCRlab activity evolved prior to the common ancestor of insects and other arthropods. In this case, the rescue of SCR-dependent labial phenotypes would be observed with the Scr orthologs outside of Insecta. The completion of a comprehensive functional analysis of Scr orthologs in Drosophila using CRISPR and RMCE enables the determination of the phylogenetic range of SCR labial and prothoracic activities and the identification of the evolutionary time point at which SCR activity changed.

2 MATERIALS AND METHODS